This application addresses important issues for understanding peptide and biogenic amine signaling in the nervous and endocrine systems. Peptide precursors are packaged into DCVs (dense-core vesicles) in the Golgi and undergo processing at the low pH in DCVs. DCVs also load biogenic amines dependent on the pH gradient. The regulated secretion of peptides and amines occurs by DCV fusion with the plasma membrane in response to elevations in Ca2+. The machinery that mediates evoked DCV exocytosis is well-characterized consisting of complexes of the vesicle v-SNARE VAMP-2 with the plasma membrane t-SNAREs syntaxin-1 and SNAP-25. SNARE complexes are assembled during DCV docking and priming reactions that precede Ca2+-triggered membrane fusion. Previous work in this project discovered CAPS (aka CADPS) as a major priming factor and revealed its activity in promoting SNARE complex assembly. Based on major advances in the previous project period, aim 1 will characterize the precise steps in the sequential SNARE complex assembly pathway that are regulated by CAPS. These consist of t-SNARE oligomer formation and the N- terminal entry of VAMP-2 into t-SNAREs. Liposome reconstitution studies will recapitulate CAPS function in DCV docking and priming. CAPS may also function on DCVs at earlier steps in the regulated secretory pathway. We discovered that CAPS localizes to DCVs in neuroendocrine cells, and that DCV localization was required for CAPS function in exocytosis. Studies on how CAPS localizes to DCVs led to identification of DMXL2 and associated proteins as potential CAPS-binding partners on DCVs. DMXL2 is a regulator of V- ATPase, and the studies of aim 2 seek to broaden the role of CAPS to several DCV functions. We will determine whether CAPS-DMXL2 interactions are direct, and whether these interactions regulate V-ATPase activity and lumenal pH of DCVs for peptide packaging in the Golgi, for prohormone conversion, and for biogenic amine uptake. The work will provide a molecular understanding for roles of CAPS on DCVs from Golgi biogenesis to plasma membrane exocytosis. Lastly, recent work revealed the first example of CAPS dysfunction in human disease. This work identified CAPS variants in individuals with early onset bipolar disorder. The mutations are in many of the functional domains on CAPS that we previously characterized. The studies of aim 3 will determine the basis for CAPS loss-of-function in early onset bipolar disorder. This work will contribute to understanding aspects of a complex polygenic disorder that may involve altered DCV-mediated BDNF secretion and/or synaptic dysfunction. Completion of the work in this application will provide a molecular description of DCV priming, fill important gaps of knowledge about the regulated secretory pathway, and determine bases for CAPS dysfunction in disease.